In this study we successfully adapted infectious HCV particles of the genotype 2a JFH1 strain to Huh7.5 cells. Adapted virus genomes retained wild-type RNA replication levels (Fig. and ) but produced 10- to 100-fold higher virus titers than the wild-type virus (Fig. and ). One major adaptive mutation (V2440L) resided at the P3 position of the NS5A-B cleavage site and reduced cleavage kinetics. This observation is in line with findings of Kim and coworkers (26
), who described that the NS3/4A protease of a genotype 1b isolate prefers valine over leucine at the P3 position. Furthermore, in none of the HCV genome sequences deposited in the European HCV database (euHCVdb) (12
) was a P3 leucine found. Apart from valine, which is the most common P3 residue, isoleucine or alanine residues are found with some isolates. Nevertheless, enhancement of virus production by reduced processing at the NS5A-B site appears to be a more general mechanism, because insertion of V2440L into three different HCV chimeras resulted in increased virus titers. Moreover, in an independent adaptation experiment with the Con1/C3 chimera, the V2440L substitution was also found (A. Kaul, I. Woerz, and R. Bartenschlager, unpublished).
Thus far, we can only speculate on how reduced NS5A-B cleavage increases virus assembly. Altered processing kinetics may affect folding and maturation of NS5A or the replicase complex. This may affect interaction of the replicase with the structural proteins, in particular core protein, during virus assembly. In this context we note that subtle differences in polyprotein processing were shown to affect the phosphorylation status of NS5A, arguing for a tight regulation of polyprotein processing and “maturation” of the cleavage products forming the replicase (28
), which in turn may affect the cross talk between the replicase and the assembly machinery.
In the first adaptation experiment, in addition to the V2440L substitution, five further mutations were detected. Three of them resided in the structural region (core, N16D; E1, I372V and I374T; and E2, I422L) and two in the NS region (NS5A, V2153A; and NS5B, V2941M). While each of the last two mutations slightly enhanced virus titers (data not shown), the mutations in the structural region had no impact on virus production when tested alone (data not shown). We observed only a slight enhancement of virus production when the I374T substitution in E1 was combined with the major adaptive mutation V2440L (mut2+5 in Fig. ). This could be traced back to the adaptation history, because both mutations coexisted in genomes isolated after the third cell passage (Table ). The I374T substitution is localized in the transmembrane domain of E1, which also is required for proper formation of E1-E2 heterodimers. This mutation could therefore play a role in the biogenesis of entry-competent E1-E2 complexes (11
). The only mutation found in NS5B (V2941M, corresponding to aa 499 of NS5B) resides in the N-terminal part of the α-helix thumb. The mutation could affect the allosteric GTP binding site (7
), but given its location on the surface of the molecule, the methionine substitution may also affect interaction with some viral or cellular factor. This assumption is affirmed by findings of Cai and coworkers. Their mutational analysis of aa 499 and other amino acids involved in GTP binding demonstrated that mutations of the GTP binding site that do not affect in vitro RdRp activity still can impair or even ablate HCV RNA replication in cell culture (9
In the second adaptation experiment, we identified three other mutations that also enhance the release of infectious virus (Fig. ) without affecting RNA replication (Fig. ). This result suggests that virus assembly is a complex process requiring numerous interactions that may be modified individually to enhance virus formation. A major adaptive mutation was identified in the first transmembrane domain of p7 (N765D). A search in the euHCVdb revealed that this residue appears to be isolate specific. While genotype 1a and 1b isolates have a glycine residue at this position, genotype 2 isolates have a serine residue, except for JFH1. Residue 765 resides in the N-terminal transmembrane α-helix of p7 and may be involved in oligomerization. According to the helical wheel diagram of that part of the protein, one can assume potential hydrophobic binding sites on the face of each helix (10
). Thus, position 765 could be involved in a hydrophobic interaction between helices 1 and 2 of adjacent p7 monomers. The major effect of this mutation was acceleration of virus release, arguing that p7 plays a very important role in virus assembly and/or release. In support of this conclusion, we and others recently found that p7 is essential for efficient assembly and release of infectious virions (23
). Whether p7 enhances the specific infectivity of virus particles is controversial (49
). At least when calculating the ratio of TCID50
/ml to extracellular core amounts, which corresponds to the specific infectivity of a virus, the N765D mutant is similar to the wild-type virus.
As we found completely different adaptive mutations in independent experiments, it is not surprising that Zhong and coworkers, who also adapted the JFH1wt isolate to a Huh7.5 subclone, found yet other mutations enhancing virus production and specific infectivity (56
). These are located in core, E2, NS3, and NS5A, and among them the G451R mutation in E2 increases infectivity titers 10-fold, whereas the other mutations have no detectable effect. In a recent study, Yi and coworkers adapted different H77/JFH1 chimeras with various crossover points between NS2 and NS3 to Huh7 cells and observed the reappearance of adaptive mutations in several independent experiments (54
). Two alternative mutations in p7 and NS3 were primarily detected, which may compensate for incompatibilities between the structural and the NS proteins that were derived from the genotype 1a H77 isolate and the JFH1 replicase. When we adapted several intergenotypic chimeras to cell culture (H77/C3, Con1/C3, and 452/C3), we also repeatedly observed, among others, chimera-specific mutations in p7 and NS3 (A. Kaul, I. Woerz, and R. Bartenschlager, unpublished). The reappearance of some mutations in independent adaptation experiments argues for limited possibilities to compensate for incompatibilities.
When we infected chimeric uPA-SCID mice with JFH1mut1-6 virus, we observed persistent replication for 15 weeks until the experiment was stopped (Fig. ). Two weeks after infection, sera of both animals contained high viral loads (about 107
HCV RNA copies/ml), which remained above 5 × 105
HCV RNA copies/ml until the animals were sacrificed. These findings are in line with results of Lindenbach and colleagues, who observed persistent replication of a J6/JFH1 chimera in uPA-SCID mice (33
). As it was recently shown that Con1 genomes carrying replication-enhancing mutations are attenuated in chimpanzees and rapidly revert to wild type (8
), in the present study we determined whether the six introduced mutations of JFH1mut1-6 were retained in the HCV genomes circulating in mice 15 weeks after infection. In contrast to the earlier study where the combination of three highly cell culture-adaptive mutations in the Con1 genome completely abolished infectivity in vivo and a genome containing only one adaptive mutation in NS5A was attenuated and rapidly reverted to wild type, in the present study we recovered mainly HCV genomes that still contained some or all of the introduced mutations (Fig. ). While this result suggests that the highly adapted JFH1 genome is viable in vivo, it is remarkable that only about half of the analyzed HCV genomes contained the major adaptive V2440L mutation in NS5A. Interestingly, in HCV genomes of animal K371L, a new mutation arose in NS5A (S2390P) that coexisted with L2440, arguing that the additional mutation may compensate for some attenuating effect of V2440L. In addition, the introduced mutation V2941M in NS5B did not revert to wild type, but in about 85% of the sequenced clones methionine was replaced by threonine. A BLAST search in the euHCVdb database revealed that most isolates possess an alanine residue at position 2941, a valine or a threonine residue is less frequently found, and none of the listed isolates posses a methionine residue at this position. Moreover, in addition to these mutations, a complex and mouse-specific pattern of nonconserved mutations was found, especially in the structural region, which will be investigated in further studies.
In summary, in this study we identified mutations in the NS region which enhance virus production and thus increase virus titers by several orders of magnitude. Our data suggest that the replicase machinery, in particular NS5A, is an important assembly determinant and that subtle alterations of polyprotein processing can have profound effects on virus production. These data will inform future studies on the molecular mechanisms underlying HCV morphogenesis.